Bioethanol Production from an Underutilized Plant, Calabash (Crescentia Cujete) Using Co-Culture of Saccharomyces Cerevisiae and Cronobacter Malonaticus

Response surface methodology (RSM) model was used to optimize ethanol production from calabash (Crescentia cujete) pulp juice using co-culture of Saccharomyces cerevisiae and Cronobacter malonaticus. The calabash pulp was squeezed with muslin cloth, and vacuum filtered to clear solution before use. The clear juice was tested for reducing sugars using the Dinitrosalicylic acid (DNS) method. Twenty three runs (23), including 3 controls, of the fermentation were conducted at varying temperatures, pH, and volumes of inoculum. The process parameters (input variables): volumes of inoculum, temperature, and pH were subjected to response surface model, using the Central composite design (CCD). Fermentation was done in conical flasks covered with cotton wool and foil in a stationary incubator for four days (96 hours). Active co-culture of Saccharomyces cerevisiae and Cronobacter malonaticus was used, with inoculum developed using Marcfaland’s method. Samples were collected every 24 hours, centrifuged, filtered and analyzed for measurement of the output variables: reducing sugar, cell density and ethanol concentration. The concentration of reducing sugars from Calabash pulp was 3.2 mg/ml. Results obtained also revealed that the fermentation can take place on a wide range of temperature; 29-31.60C . The optimal pH range for performance of the co-culture for the fermentation process was pH range 7.9- 8.0. The optimum volume of inoculum was 5.5%v/v (ie 5.5 ml in 94.5ml juice). The optimized process using the RSM model gave 6.97% v/v bioethanol at 29oC and pH 7.9. The bioethanol yield from Calabash substrate is reasonable with co-culture considering the concentration of reducing sugars obtained from the juice and the duration of the fermentation.